Overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated in the[unreadable] pathogenesis of airway inflammation of asthma. The long-term goal of this research is to understand the[unreadable] regulation of iNOS activity and to devise novel methods to regulate it. Although much is known about[unreadable] factors affecting the synthesis and catalytic activity of iNOS, little is known about its cellular regulation. We[unreadable] have recently shown that iNOS is degraded through the ubiquitin-proteasome pathway. The specificity of[unreadable] the ubiqutination system is mainly provided by the specific ubiquitin ligase enzyme (E3) that recognizes and[unreadable] binds to the target protein. Our preliminary data identified an F-box-containing protein; we termed UBLinos[unreadable] that is a likely candidate to be the E3 ubiquitin ligase for iNOS. Additional preliminary data suggest that cells[unreadable] regulate NO synthesis by temporal and spatial regulation of iNOS. These mechanisms include a relatively[unreadable] rapid rate of iNOS turnover and sequestration of iNOS to a perinuclear location we termed the "physiologic[unreadable] aggresome." Furthermore, we have recently discovered that a specific class of imidazole like compounds[unreadable] acts by co-translational inhibition of iNOS assembly. Thus, the use of these compounds represents a[unreadable] specific and an efficient method to inhibit iNOS production.[unreadable] We propose to test the following hypotheses: A) An F-box-containing protein (UBLinos) is the E3 ligase for[unreadable] iNOS and it plays a central role in iNOS cellular regulation. B) The cellular temporal and spatial regulation[unreadable] of iNOS plays a critical role in the observed upregulation of iNOS associated with airway inflammation.[unreadable] iNOS translational inhibitors will reduce iNOS levels and ameliorate inflammation in experimental mouse[unreadable] models of asthma. To test these hypotheses we propose studies with the following specific aims: 1)[unreadable] Determination of mechanisms of iNOS regulation by UBLinos. 2) Elucidation of the cellular temporal and[unreadable] spatial regulation of iNOS in airway inflammation. 3) Evaluation of the use of iNOS translational inhibitors in[unreadable] experimental asthma by testing their effects in mouse models of asthma. The above studies will be done in[unreadable] cultured cells as well as in primary cells obtained from normal subjects, patients with asthma and from[unreadable] mouse models of asthma. The results of these studies will enhance our understanding of the regulation of[unreadable] NO synthesis by iNOS in airway inflammation and lay the groundwork for therapeutic strategies aimed at[unreadable] regulating iNOS in such disorder.[unreadable]